Treatment of a pathological fibrinolytic state in patients

ABSTRACT

THE COMPOUNDS 4-AMINOMETHYLBICYCLO-(2.2.1)-HEPTANE1-CARBOXYLIC ACID, 4-AMINOMETHYLBISOCO-i2.2.2)-OCTANE-1CARBOXYLIC ACID 5-AMINOMETHYLBICYCLO-(3.2.2)-NONANE-1CARBOXYLIC ACID AND THE CORRESPONDING 2,5 AND 6,8-DIKETO COMPOUND RESPECTIVELY ARE USEFUL IN AN-FIBRINOLYTIC COMPOUNDS.

United States Patent 3,720,775 TREATMENT OF A PATHOLOGICAL FIBRINOLYTIC STATE IN PATIENTS Larry J. Loeiller, North Wales, Pa., assignor to Merck & Co., Inc., Rahway, NJ.

No Drawing. Application June 13, 1969, Ser. No. 833,161,

now Patent No. 3,634,499, which is a continuation-inpart of abandoned application Ser. No. 690,404, Dec. 14, 1967. Divided and this application June 25, 1971, Ser. No. 157,035

Int. Cl. A61k 27/00 US. Cl. 424319 7 Claims ABSTRACT OF THE DISCLOSURE The compounds 4-aminomethylbicyclo-[2.2.1]-heptanel-carboxylic acid, 4-aminomethylbicyclo-[2.2.2]-octane-1- carboxylic acid, S-aminomethylbicyclo-[3.2.2 ]-nonane lcarboxylic acid and the corresponding 2,5 and 6,8-diketo compound respectively are useful in anti-fibrinolytic compounds.

This application is a division of US. application Ser. No. 833,161 filed June 13, 1969, now US. Pat. No. 3,634,499 which was in turn a continuation-in-part of US. application Ser. No. 690,404 filed Dec. 14, 1967, now abandoned.

This invention relates to new anti-fibrinolytic compounds and to a method of counteracting certain hemorrhagic conditions and other disorders resulting from a patho logical fibrinolytic state in patients. More specifically, this invention relates to new compounds of the structure in which n is 1, 2 or 3 and in which X is either H or 0. More specifically also, it relates to the prevention or treatment of a pathological fibrinolytic state in patients by the oral administration of from 1 to 20 and preferably 2 to 8 mg./kg. of body weight per day of the above compounds for varying periods of treatment.

The dissolution of fibrin deposits in mammals is due to their lysis by the enzyme plasmin (fibrinolysin) which is formed in the blood from plasminogen, also present in the blood. This conversion from plasminogen to plasmin is promoted by activators in the blood and it would appear that excessive fibrinolytic activity results from an overabundance of such activators. When too much plasmin is present, the clotting system of the blood becomes unbalanced, viable clots cannot be maintained, and hemorrhage may result. This situation is known as a fibrinolytic state. Other enzyme systems (i.e., the kallikreins, complement) may also be activated in an undesirable manner when such a state exists.

An interest has recently developed in anti-fibrinolytic agents, i.e. drugs which will inhibit the activation of plasminogen to form plasmin. These anti-fibrinolytic agents are believed to interfere with the function of the activators of converting plasminogen to plasmin. The clinical uses of such drugs include their administration to persons undergoing various kinds of surgery (such as heart-lung and prostate surgery), obstetrical hemorrhage problems,

3,720,775 Patented Mar. 13, 1973 menorrhagia, and many other uses which have been suggested in the literature (e.g. see Nilssen, Acta Medica Scand, Suppl. 448, volume 180, 1966).

A standard anti-fibrinolytic agent, against which newer ones are generally tested and compared is epsilon aminocaproic acid, known as EACA. One deficiency of this agent has been the very high dosages needed; in some cases 3-6 grams or more every 4 to 6 hours. Also, side effects such as dizziness, nausea and diarrhea have been observed. More recently, two more potent agents have been described, namely trans-4-aminomethylcyclo hexane carboxylic acid (AMCHA) and 4-aminomethylbenzoic acid (PAM BA). Each is reported to be more active than EACA by both in vitro and in vivo tests (e.g. see Anderssen et al. Scand. J. Haemat (1965 2, 230 and Melander et al. Acta Pharmacol et Toxicol 1965, 22, 340, both of which discuss AMCHA).

I have found a class of aminomethyl bicyclic carboxylic acids which shows activities of 5 to 50 times that of EACA in tests essentially the same as those known to correlate with clinical results. I have thus also found an improved anti-fibrinolytic method of therapeutic treatment requiring much smaller doses of the drug.

The new compounds of my invention have the general structure in which n is 1, 2 or 3 and X is H or 0. They are thus 4-aminomethylbicyclo- [2.2. l -heptanel -carboxylic acid, 4-aminomethylbicyclo-[2.2.21-octane 1 carboxylic acid, 5 aminomethylbicyclo-[3.2.2]-nonane-1-carboxylic acid and the corresponding diketo compounds. Further modification of this molecular arrangement, such as substitution on the nitrogen or deletion of the -CH between the nitrogen and the ring, appears to destroy the activity.

Especially preferred is the compound 4-aminomethylbicyclo-[2.2.2]-octane-1-carboxylic acid, which shows at least 50 times the activity of EACA in vitro and is 8 times as active in vivo in dogs. This compound has the further advantage of being well absorbed by oral administration; almost as well as when given intravenously.

The compounds of this invention are prepared by catalytic reduction of the corresponding 4-cyanobicycloheptane or octane or 5-cyanobicyclo-nonane-carboxylic acid. In the case of the bicycloheptane and bicyclooctane acids, the cyano compound is known [Roberts et al. J. Am. Chem. Soc. 75, 637 (1953)] and [Wilcox et al. Journal of Organic Chemistry, 33, 877 (1968)]. The corresponding bicyclononane cyano acid is prepared in a similar synthetic route in which 1,3-dibromopropane is substituted for 1,2-dibromoethane in the second step. These syntheses are described in the Flow Sheet. The keto compounds are similarly prepared except that the keto groups are protected until the end as ethylene ketal derivatives. The carboxylic esters are prepared by direct esterification of the amino acids such as by the use of alcoholic hydrogen chloride or thionyl chloride followed by alcohol. Similarly, the alkanoyl amino compounds are prepared by acylation of the amino acids. These esters and alkanoyl amino derivatives are to be considered as included within the scope of the compounds represented by the above structural formulae and in the appended claims.

FLOW SHEET 1 l? 2 E R000 000R moo-@0001:

Hz-COOR nooo@ -coon ROOC clamn coon ROOC@-COOR H000 (CH2), COOR nooo@ooon A NC@COOR mum-@420 OR A L l 1 n 1(8) nc@ ooon NC@COOH J 1(8 lo nc@ooon Havana-212 coon Nmom@ ooon HzN-CH: (0112).. c0011 Equivalents: R=ethyl; n=2 or 3.

Reactions 1) Claisen condensation with NaOCzHs.

2) Reaction with NaH or other strong base followed by BR(CH2)nB1.

(3 Reaction with HS(CH2)2SH in presence of BFa etherate in glacial acetic acid.

Reduction with Raney nickel. 5) Gentle saponification with a base such as NaOH in aqueous alcohol. Reaction with triethylamine and chloroformic ester, followed by ammonia to convert the anhydride to the amide. Dehydration with POCla in 1,2-dichloroethane or pyridine. Heat with base such as NaOH in aqueous alcohol. Catalytic reduction, such as over Pt in aqueous alcoholic H01 solution 0) Reaction with 1 Z-ethylene glycol in presence of toluene sulfonic acid in benzene. 1 Heating with acid.

The compounds of this invention are used in the method of this invention by either oral or intravenous administration, although the oral route is preferred. The esters and amides of this class of compounds are not them selves very active in vitro but the action of enzymes in vivo may cause the slow liberation of the highly active amino acids, thus providing a prolonged availability of the drug in the body. This is important because of the tendency of these drugs to be swiftly eliminated in the urine.

The compounds of this invention can be used in any pharmaceutically acceptable carrier, in the form of pills, tablets or capsules. The pharmaceutically acceptable salts (both of the amino groupsuch as the hydrochloride, hydrobromide, sulfate, citrate, tartrate, etc.-and of the carboxy groupsuch as the alkali metal, alkaline earth metal, etc., salts) are readily usable, especially in injectable compositions.

The invention can be illustrated by the following ex amples.

EXAMPLE 1 4-aminomethylbicyclo-[2.2.21-octane-l-carboxylic acid (A) Hydrochloride salt.-To a solution of 2.80 g. (0.0156 mole) of 4-cyanobicyclo-[2.2.21-octane-1-carboxylic acid (Roberts et al. JACS 1953, 75 637) in 100 ml. ethanol was added 5.0 ml. 6 N hydrochloric acid and 500 mg. platinum oxide. During hydrogenation on a Parr apparatus at room temperature and 40 lbs/in. pressure, the theoretical quantity of hydrogen was absorbed during the first half hour. After 2 hours, the hydrogenation was stopped and the reaction mixture filtered through sintered glass to remove the platinum catalyst. Evaporation of the clear, colorless filtrate in vacuo (30-40 C.) left a white solid which was reevaporated with three portions of 90% ethanol to remove most of the excess hydrochloric acid. Purification was accomplished by dissolution in 200 ml. hot 95% ethanol and reprecipitation with 750 ml. absolute ethyl ether. The white solid, M.P. 318-319" C. dec. (placed in sealed capillary at 250 C.) after air drying, was obtained in 90.5% yield. Three recrystallizations from 95% ethanol ether gave analytically pure material, M.P. 318 319 C. dec., which was dried at 110 C. over phosphorous pentoxide for 18 hours at 0.08 mm. Hg. The product exhibited a single circular ninhydrin positive spot (pink) upon thin layer chromatography on silica gel with 3:1:1 n-butanol, acetic acid, water R =0.65.

(B) Free amino acid.--A sample of analytically pure hydrochloride (300 mg.) was dissolved in 2 ml. distilled water, placed above a column of water-washed Dowex-l acetate (20 g. wet weight) and eluted with distilled water. Tests with ninhydrin indicated that all of the free amino acid was contained in the first few ml. of eluant. The first 100 ml. of eluant, when evaporated in vacuo (60-70 C.), afforded the free amino acid. After one to three recrystallizations from water-acetone, the air-dried material or material dried at 110 C. for 24 hours melted at 269- 274 C. with decomposition and some darkening and softening at 265 C. (placed in sealed capillary at 180 C.), and the material analyzed slightly low in carbon. Redrying at 137 C. for 24 hours at 0.35 mm. Hg over P raised the melting point to 280-283" C. dec. (softening at 274.5).

EXAMPLE 2 (A) Diethyl-6,8-dioxobicyclo-[3.2.2]-nonane-1,5- dicarboxylate To 250 ml. of 1,2-dimethoxyethane (freshly distilled over sodium hydride) in a dry atmosphere under nitrogen was added 25.0 g. of a 55.7% dispersion of sodium hydride in mineral oil (13.92 g. sodium hydride) (0.58 mole). To this was added in portions, over a half-hour period, with mechanical stirring, 73.8 g. (0.29 mole) finely powdered diethyl-1,4-cyclohexanedione 2.5 dicarboxylate [Org Synth. 45, 25 1966)]. Hydrogen was evolved, the mixture became slightly warm, and a pink suspension resulted. After removal of ml. of 1,2-dimethoxyethane by distillation, there was added 370 g. (1.84 mole) 1,3 dibromopropane. After removal of most of the remaining 1,2 dimethoxyethane by distillation, the mixture was stirred and heated at 120-130 C. in an oil bath for 65 hours. The solids were removed by filtration, washed with 3X 20 ml. chloroform, and diluted with 400 ml. hexane. Filtration, washing with hexane and air drying left 30.0 g. (35%) crystals, M.P. -133". (Reported: 132 C.) Guha et al., Chem. Ber. 1392 (1939). Before use in the next synthetic step, the material was recrystallized from 95 ethanol (high recovery) and was dried overnight at 50 C. in vacuo, M.P. 130.5-133" C.

(B) Diethyl-6,8-bis-ethylenedithiobicyclo- 3 .2.2 nonane-1,5-dicarboxylate To a mixture of 18.30 g. (0.062 mole) finely powdered diethyl 6,8 dioxobicyclo [3.2.2] nonane 1,5 dicarboxylate and 17.52 g. (0.186 mole) 1,2 ethane dithiol was added 22.0 g. (0.155 mole) boron trifluoride etherate. The resultant mixture was stirred at room temperature with protection from moisture for three days. After the addition of ml. chloroform, the mixture was extracted with 3x 60 ml. water, 4 50 ml. 5% sodium hydroxide, then 3X 60 ml. water. After drying over magnesium sulfate, evaporation of the solvent left a viscous oil. Trituration with 70 ml. of methanol and chilling afforded a white solid, M.P. 101-103.5 C., 17.6 g. (63.2%). Recrystallization from ethanol, then two recrystallizations from n-hexane gave analytically pure material as needles, M.P. 103.5105 C.

(C) Diethyl bicyclo-[3.2.2]-nonane-1,5-dicarboxylate A solution of 10.70 g. (0.0238 mole) diethyl-6,8-bisethylenedithio bicyclo-[3.2.2]-nonane-1,5 dicarboxylate in 300 ml. 95% ethanol was refluxed with 300 g. (wet weight) active Raney nickel catalyst for four days. After removal of the nickel catalyst by filtration, the ethanol was removed in vacuo and the product separated from the two-phase mixture with water by extraction into ether. The combined ether layers were dried over anhydrous magnesium sulfate, filtered and stripped, giving 5.65 g. colorless liquid (88% crude). A repeat preparation gave the product in 95% crude yield.

Vacuum distillation of 14.5 g. crude material afforded 13.1 g. colorless liquid, B.P. 142-145 C./0.8 mm. Hg. Two repeat distillations gave a sample for analysis, B.P. 114-115" C./0.05 mm. Hg.

Vapor phase chromatography of this material indicated the presence of a minor contaminant (5% or less), not removable by distillation. However, the material obtained after one distillation proved of purity sufficient for use in the preparation of ethyl hydrogen bicyclo- 3.2.2]-nonane-1,5-dicarboxylate.

(D) Ethyl hydrogen bicyclo-[3.2.2]-nonane-1,5- dicarboxylate To a solution of 0.97 g. (0.042 mole) clean sodium spheres in 50 ml. absolute ethanol was added 5.0 ml. distilled water and 11.27 g. (0.042 mole) diethyl bicyclo- [3.2.2]-nonane-1,5-dicarboxylate in one portion. The mixture was stirred magnetically and refluxed for 20 hours under nitrogen. After removal of most of the ethanol in vacuo from the suspension, 50 ml. water was added to the residue. Unsaponified diester was removed by extraction into 3x 25 ml. ether, and 1.81 g. (16.1%) ester was recovered after drying and removal of the ether. Acidification of the chilled aqueous layer with 8 ml. 6.0 N hydrochloric acid gave a white solid which was extracted into 4x 50 ml. ethyl acetate. The combined ethyl acetate layers, after drying, afforded 7.16 g. viscous oil. Dissolution in 100 ml. chloroform and chilling enabled removal of 0.63 g. (6.5%) unwanted diacid; however, the separation could not be cleanly and completely achieved in this manner, as monitored by TLC studies. Evaporation of the filtrate and chromatography over silica gel (150 g.), using chloroform as the eluant, gave the mono acid while the diacid did not elute from the column.

There was recovered 6.36 g. (62%) of a slightly oily solid which appeared homogenous by TLC. Recrystallization from hexane (Dry Ice-acetone) gave material melting at 5153.5 C. Further recrystallization of a small quantity and finally sublimation produced an analytical sample, M.P. 53.555 C.

(E) Ethyl S-carboxamidobicyclo-[3.2.21-nonane-1- carboxylate In a dry nitrogen atmosphere, 3.96 g. (0.0165 mole) ethyl hydrogen bicyclo [3.2.2]-nonane-l,S-dicarboxylate was dissolved in 60 ml. dry (4A sieves) chloroform. After cooling to 10 C., 1.67 g. (0.0165 mole) triethylamine was added, then over a 10 minute period 1.79 g. (0.0165 mole) ethyl chloroformate in 10 ml. chloroform with stirring, keeping the temperature between 10 and C. After stirring for an additional 15 minutes at 5 C., ammonia gas was bubbled through the stirred mixture for 15 minutes with continued cooling. The ice-bath was removed and the suspension allowed to stir for 17 hours. After a thorough washing with water, 0.5 N hydrochloric acid, saturated sodium bicarbonate, water and drying over magnesium sulfate, removal of the chloroform in vacuo left a white solid, which was shownto be homogenous by thin layer chromatography; M.P. 80.5-83 C., yield 2.83 g. (71.6% Three recrystallizations of a small sample from benzene-hexane gave an analytical sample (needles), M.P. 83.585 C.

(F) Ethyl 5-cyanobicyclo-[3.2.2]-nonanel-carboxylate To 1.44 g. (6.0 mm.) ethyl 5 carboxamidobicyclo- [3.2.2]-nonane-l-carboxylate dissolved in 30 ml. freshly distilled (4A molecular sieves) 1,2-dichloroethane was added 2.60 ml. phosphosous oxychloride. The mixture was refluxed for 20 minutes with protection from moisture. After removal of most of the excess solvent and phosphorous oxychloride by distillation in vacuo (60-70" C.), the red liquid residue was treated with cold saturated sodium bicarbonate and the product extracted into n-pentane. After washing with sodium bicarbonate and water, and

drying over magnesium sulfate, evaporation of the pentanelayer afforded a pale yellow oil, 1.13 g. (86% crude). The material was shown to be homogenous by thin layer chromatography, possessed the expected absorptions in the infrared and was saponified directly to the corresponding cyano acid without further purification.

(G) S-cyanobicyclo-[3.2.2]-nonane-1-carboxylic acid To 1.00 g. (4.52 mm.) ethyl-S-cyanobicyclo-[3.2.2]- nonane-l-carboxylate dissolved in 20 ml. absolute ethanol was added 2.70 ml. of 2.00 N sodium hydroxide. The mixture was refluxed for 2 hours, then stirred over night at room temperature. After removal of the ethanol in vacuo, the remaining solid was dissolved in 20 ml. water, extracted with ether, then acidified with hydrochloric acid and the product extracted into ether. After washing and drying over magnesium sulfate, removal of the ether afforded 790 mg. white solid, M.P. 184-188" C. (87% crude yield). The material was shown to be homogeneous by thin layer chromatography. The material, upon recrystallization from benzene, remained unchanged in melting point, and was found to trap large quantities of solvent removed only on drying in vacuo at 80 C. A small sam ple was recrystallized three times from bezene and after drying for analysis, melted at 186188 C.

(H) S-aminomethylbicyclo-[3.2.2]-nonane-l-carboxylic acid hydrochloride To a solution of 380 mg. (1.96 mm.) S-cyano bicyclo- [3.2.2]-nonane-l-carboxylic acid in 40- ml. absolute ethanol was added 20 ml. distilled water, 2.0 ml. 6.0 N hydrochloric acid, and 200 mg. platinum oxide. The material was hydrogenated on a Parr apparatus at room temperature was refluxed for 2 hours, then stirred overnight at catalyst by filtration, the filtrate was stripped in vacuo at 60-70 C., then reevaporated several times with fresh portions of distilled water. A White solid remained, M.P. 286-289 C., in nearly quantitative yield. Thin layer chromatography indicated one ninhydrin positive spot. One recrystallization from 95% ethanol-ether afforded the pure product, M.P. 291.5-293 C. dec., 371 mg. (81%). The sample was recrystallized three more times from ethanolether for analysis, without change in melting point.

EXAMPLE 3 (A) Diethyl-Z,S-bis-ethylenedioxobicyclo- [2.2.2.]- octane, 1,4-dicarboxylate Diethyl-2,5-bis-ethylenedioxobicyclo [2.2.2] octanel,4-dicarboxylate was prepared from the corresponding dioxo compound essentially as described by H. D. Holtz and L. M. Stock, J. Am. Chem. Soc. 86, 5183 (1964).

(B) DL-ethyl hydrogen-2,5-bis-ethylenedioxobicyclo- [2.2.2] -octane-1,4-dicarboxylate To a solution of 58.10 g. (0.157 mole) diethyl-2,5- ethylenedioxobicyclo-[2.2.2]-octane 1,4 dicarboxylate in 900 ml. ethanol was added 78.5 ml. of 2.00 N NaOH. The mixture was stirred magnetically and refluxed under nitrogen for 22 hours. After removal of most oflthe ethanol in vacuo, 250 ml. water was added and the mixture extracted with ethyl acetate for recovery of 20% of the starting diester which could be recycled. The chilled aqueous layer was acidified with 30 ml. of 6 N HCl, then extracted several times with ethyl acetate. The combined ethyl acetate layers were Washed with water, dried over magnesium sulfate, filtered, and the solvent stripped in vacuo, affording 38.4 g. of white powder, which was shown by thin layer chromatography to be a mixture of the desired mono acid with diacid (R s of 0.67 and 0.41 respectively on silica gel, employing :25 :4 benzene-dioxane-acetic acid as the eluant). Passage of this material (66.5 g.) through a column of 500 g. of silica gel, employing chloroform and 9:1 chloroform ethyl acetate as the eluant enabled clean separation of the mono and diacids. Recrystallization of the crude mono acid from the column (52.2 g.) from benzene afforded the pure product, M.P. 139-141 C., in 58% yield based upon unrecovered diacid. Several recrystallizations from %1 ethanol gave an analytical sample, M.P. 140.5142 C.

(C) DL-ethyl-4-carboxamido-2,5-bis-ethylenedioxobicyclo-[2.2.2]-octane-l-carboxylate To a solution of 34.23 g. (0.10 mole) ethyl hydrogen- 2,5-bis-ethylenedioxobicyclo [2.2.21 octane-1,4-dicarboxylate (2) in 500 ml. dry chloroform was added 20.24 g. (0.20 mole) triethylamine. To the stirred, chilled (10 C.) solution was added 11.40 g. (0.10-5 mole) ethyl chloroformate over a 30-minute period, maintaining the temperature at -10 C. After the addition was complete, stirring was continued at this temperature for an additional thirty minutes. With continued cooling in an ice-salt bath, ammonia gas was bubbled into the mixture for 2025 minutes, the mixture allowed to warm to room temperature with stirring overnight. After removal of the chloroform in vacuo, addition of water and thorough extraction with ethyl acetate afforded nearly analytically pure amide (25.2 g., 74% yield), M.P. 159.5-161.5 C. Three recrystallizations from 95% ethanol gave an analytical sample, M.P. 160.5-162 C.

9 (D) DL-ethyl-4-cyano-2,5-bis-ethylenedioxobicyclo- [2.2.2]-octane-1-carboxylate To a solution of 11.30 g. (0.033 mole) of ethyl-4-carboxamido 2,5 bis-ethylenedioxobicyclo-[2.2.2]-octane l-carboxylate in 170 ml. dry pyridine was added slowly 17 ml. phosphorous oxychloride. The resulting solution was heated at 70-80" C. for about 1 hour. The volume was reduced to about 30 ml. by distillation in vacuo and the cooled residue added cautiously to about 300 ml. saturated sodium bicarbonate containing ice. Extraction with ethyl acetate, drying over magnesium sulfate, filtering and stripping in vacuo left a yellow oil which solidified when chilled. Recrystallization from hexane afforded pure product, M.P. 8284 C., yield 9.0 g. 84% Recrystallization from hexane gave an analytical sample, M.P. 82.5-84.

(E) DL-4-cyano2,S-bis-ethylenedioxobicyclo-[2.2.2]- octane-l-carboxylic acid To 2.43 g. (0.075 mole) ethyl 4 cyano-2,5-bis-ethylenedioxobicyclo-[2.2.2]-octane-l-carboxylate dissolved in 50 ml. ethanol was added 8.0 ml. 2.0 N sodium hydroxide. The mixture was refluxed for 6 hours under nitrogen, then left overnight. After removal of the ethanol in vacuo and the addition of water, 0.31 g. (12.7%) starting material was recovered by ether extraction. Careful acidification of the cold aqueous layer produced an oil which quickly began to solidify. The product was collected either by filtration or extraction into ethyl acetate. After one recrystallization of the crude cyano acid (1.80 g., 93% based on unrecovered ester), 1.47 g. of pure product was obtained, M.P. 223.5-225" C. Recrystallization from acetonitrile gave an analytical sample, M.P. 223.5-225 C.

(F) DL-4-aminomethylbicyclo- [2.2.2] -octa-'2,5- dione-l-carboxylic acid To 590 mg. (2.0 mm.) 4-cyano-2,S-bis-ethylenedioxobicyclo-[2.2.2]-octa-2,5-dione-l-carboxylic acid in 40 ml. ethanol was added 4.0 ml. of 1.0 N hydrochloric acid and 100 mg. platinum oxide. The compound was hydrogenated for 2 hours at 30 lb./in. filtered and evaporated, then refluxed for 8 hours with 20 ml. 6 N hydrochloric acid. After evaporation in vacuo and re-evaporation several times with small portions of water, the crude amino acid hydrochloride was dissolved in ml. water and passed through a column of Dowex-l acetate (20 g.). Evaporation of the eflluent gave the crystalline product, M.P. 360 C. Several recrystallizations from water-acetone gave the amino acid for analysis (147 mg), M.P. 360 C. The material appears to darken upon heating above 100 C., exhibits the expected infrared absorptions (1720 cm? (C=O); 1610 cm. (COOH); 1560 cm.- (NI-l 1450 cmr 1380 cmr- 1270 cm. 1210 cmf 1140 cm. 780 cmr 560 cmf 350 cmf and appears homogenous upon thin layer chromatography [R =0.40 (ninhydrin) on silica gel with 3: 1:1 butanol-acetic acidwater].

It will be observed that the diketo compounds of Example 3 have been identified as having D and L configurations due to a lack of a plane of symmetry, i.e. their mirror images are not superimposable.

EXAMPLE 4 (A) 4-cyanobicyclo-[2.2.1]-heptane-1-carboxylic acid The above compound was prepared according to the procedure reported by Wilcox et al. Journal of Organic Chemistry 33 (1968), page 877.

(B) 4-aminomethylbicyclo-[2.2.l]-heptane-1- carboxylic acid hydrochloride To mg. (1.0 mm.) of 4-cyan0bicyclo-[2.2.1]- heptane-l-carboxylic acid dissolved in 20 ml. ethanol was added 8 ml. of water, 2.0 ml. of 1.00 N hydrochloric acid and 100 mg. platinum oxide. The compound was hydrogenated on a Paar apparatus at 35 lbs/in. at room temperature for one hour. After removal of the catalyst by filtration through sintered glass, the fitrate was evaporated in vacuo at steam bath temperature. It was then washed three times with fresh 10 ml. portions of 95% ethanol. A white solid remained, mg. (90%) with an M.P. 255- 25 8 C. with decomposition. The material appeared to be homogenous by thin layer chromatography [R =0.38 (ninhydrin)-silica gel with 3:1:1 butanol-acetic acidwater]. Three recrystallizations from ethanol-ether gave an analytical sample, M.P. 256258 C. with decomposition.

EXAMPLE 5 Compounds are tested in vitro by measuring the effect of the inhibitor at various concentrations on the lysis times of a fibrin clot with a constant concentration of streptokinase in plasminogen-rich plasma. The concentration of the inhibitor which increases the geometric mean lysis time by 50% is estimated. Epsilon amino caproic acid (EACA) is used as a standard and the relative potencies are obtained, with the following results:

Relative in vitro activity Compound: (weight basis) (A) Reference compound: EACA 1.0 (B) New compounds:

4 aminomethylbicyclo [2.2.2]-octane-1- carboxylic acid 53.7 5 aminomethylbicyclo [3.2.2.1-nonanel-carboxylic acid 10.8 DL-4-aminomethylbicyclo [2.2.2]-octa- 2,5-dione-1-carboxylic acid 8.0

I claim:

1. A method of counteracting a pathological fibrinolytic state in patients which comprises the intravenous or oral administration of from 1 to 20 mg/kg. of a compound selected from the group consisting of HgN--Ol[g-C( CH1) 04: 0 o rt CHz-C [l and the pharmaceutically acceptable salts thereof, in which n is 1, 2 or 3 and X is either H or O.

2. The method of claim 1 in which the compound used It is 2 and X is H 3. The method of claim 1 in which in the compound used It is 1 and X is H 4. The method of claim 1 in which n is 3 and X is H 5. The method of claim 1 in which n is 2 and X is O. 6. The method of claim 1 in which n is 3 and X is O. 7. The method of claim 1 in which daily administration is from 2 to 8 mg./kg. of body weight.

References Cited UNITED STATES PATENTS 3,457,347 7/1969 Rubricius 424-319 SAM ROSEN, Primary Examiner 

